GSM7213605: HEK293, pCAG-TWIST1, TWIST1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
TWIST1
Cell type
Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
HEK293
cell type
Human embryonic kidney
cell line
HEK293
genotype
WT
clone
--
treatment
transfection with pCAG-TWIST1
time
24h
spike-in cell_type
--
spike-in cell_line
--
antibody
TWIST1 (Abcam, #ab50887)
reference genome_for_alignment
hg38
reference genome_for_processed_files
hg38
geo_loc_name
missing
collection_date
missing
Sequenced DNA Library
library_name
GSM7213605
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells (about 1 confluent 10-cm plate or ~10-20 million cells) were crosslinked with 1% methanol-free formaldehyde (Pierce, 28908) in PBS for 10 min at room temperature and then quenched by adding 2.5 M glycine to 125 mM final concentration and incubating for 10 min. Cells were washed in PBS with 0.001% v/v Triton X-100, harvested by scraping, and collected by centrifugation for 5 min at 4°C. Cells were washed with PBS and flash frozen for storage at -80°C. Cell pellets were later thawed on ice for 30 min, and then sequentially resuspended in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 0.25% Triton X-100, 1x cOmplete EDTA-free protease inhibitor cocktail (PIC), 1 mM PMSF), lysis buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x cOmplete EDTA-free protease inhibitor cocktail, 1 mM PMSF), and lysis buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1x PIC, 1 mM PMSF), with 10 min incubations in each buffer, with rotation. Lysates were sonicated for 10-15 cycles of 30s ON/30s OFF on high power using the Bioruptor Plus (Diagenode), then diluted in additional lysis buffer 3 and clarified by centrifugation for 10 min at max speed at 4°C. Triton X-100 was added to 1%, and a small aliquot was used to extract DNA to check chromatin yield and size distribution, by dilution in elution buffer (1% w/v SDS and 100 mM NaHCO3) and incubation with 200 mM NaCl and RNase A (Thermo, EN0531) at 65°C for 1 h, then proteinase K (Thermo, EO0491) at 65°C for 1 h, and clean up with the ChIP DNA Clean & Concentrator-5 kit (Zymo, D5205). DNA was quantified by Qubit dsDNA high sensitivity kit, and the remaining chromatin was then normalized for immunoprecipitations. For TWIST1 acute depletions, chromatin from O9-1 mouse CNCCs were added prior to ChIP at ~10% of the total chromatin as a spike-in control. For H3K27ac, 5 ug of antibody was used per ChIP; for TFs, 9 ug of antibody was used per ChIP, except for dissected mouse embryos where 4.5 ug was used in half of the total ChIP volume. ChIPs were incubated overnight, then incubated for 4-6h with 100 ul Dynabeads Protein A (Invitrogen, 10002D) or Protein G (Invitrogen, 10004D) prewashed with 0.1% w/v BSA in PBS, then washed 5x with RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% Igepal CA-630, 0.7% w/v sodium deoxycholate), once with 50 mM Tris-HCl pH 8, 10 mM EDTA, 50 mM NaCl, and eluted in elution buffer at 65°C for 30 min. Eluate was then reverse crosslinked and treated with RNase A and proteinase K, and then DNA was extracted with the ChIP DNA Clean & Concentrator-5 kit. Libraries were prepared using the NEBNext Ultra II DNA kit (New England Biolabs, E7645S) using up to 50 ng of input or ChIP DNA, with ~4-8 cycles of amplification, with no pre-PCR size selection but a post-PCR double-sided 0.5x/0.9x Ampure XP bead clean-up.